The subject matter of the published European patent application No. 0 004 913, corresponding to U.S. patent application Ser. No. 29,415, filed Apr. 12, 1979, is, among others, a process for the preparation of 17-C-steroid-.alpha.-propionic acid compounds, particularly for the preparation of 3-oxo-pregna-4-en-20-carboxylic acid (.DELTA..sup.4 -BNC) and/or 3-oxo-pregna-1,4-dien-20-carboxylic acid (.DELTA..sup.1,4 -BNC) by microbial side-chain splitting of 17-C-side-chain steroids. The process is characterized in that defect mutant microorganisms which give steroid compounds with the 17-C-.alpha.-propionic acid substituent even in the absence of inhibitors inhibiting either steroid ring cleavage and/or microorganism growth are grown in an aqueous nutrient medium under aerobic conditions in the presence of the steroid substrate with enrichment of the 17-C steroid-.alpha.-propionic acid compounds in the fermentation broth and .DELTA..sup.4 -BNC and/or .DELTA..sup.1,4 -BNC thus formed are isolated.
It is preferable to work with defect mutant microorganisms which have been grown by mutation and subsequent selection from previously selected wild strains which can grow on steroid compounds with 17-C-side chains as the preferable sole carbon source with an at least about equal splitting rate for the side chains as for the cleavage rate of the ring portion of the steroid compounds, but preferably an increased side-chain splitting rate. Particularly the block mutants from wild strains have been employed which, when grown on steroid compounds, yield a selective splitting under standard conditions according to the general formula: EQU I=a.multidot.10.sup.b
where a denotes the growth factor and b the selectivity factor of the wild strain growth, and the selectivity index I of the wild strain is at least particularly 10.sup.5. The selectivity factor b of the wild strain used for growing the defect mutants should be at least 2, and the growth factor a preferably at least 0.2, particularly at least 1.
The growing of the wild strains of the subsequent selection according to their selectivity index I is effected on a 17-C-side-chain steroid as a carbon source of the type which is used as a starting material in decomposition methods, this steroid compound being preferably used as the sole carbon source for growing the wild strains.
The defect mutants preferably are strains which do not or practically do not grow on a mutant-separating medium in the growing of the mutant population from a known mutation of the selected wild strains, while the undesired accompanying mutant strains grow and were thus killed during their growth. Preferably block mutants of the genera Achromobacter, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacterium, Serratia or Streptomyces are used.
The starting steroid compounds are those with saturated and/or unsaturated 17-C-side chains, where the side-chain radicals have preferably up to 10 carbon atoms, particularly 8 to 10 carbon atoms. Particularly suitable as starting materials are sterols of animal or vegetable origin, particularly cholesterol, sitosterol, stigmesterol, campesterol and/or ergosterol or their derivatives, such as cholestenone, sitostenone or stigmastenone.
Another embodiment of this process for the preparation of .DELTA..sup.4 -BNC and .DELTA..sup.1,4 -BNC is described in the published European patent application No. 0 015 308, which corresponds to U.S. patent application Ser. No. 128,223, filed Mar. 7, 1980. In this application defect mutants were used which were obtained, however, from a wild strain which supplies, at least partly, 17-C-steroid-.alpha.-propionic acid compounds in the aerobic growth on sterol compounds in the presence of inhibitors for the enzymatic ring cleavage of the sterol compounds. In particular, those wild strains were isolated and grown which show, in the growth on sterol compounds with saturated or unsaturated alkyl radicals on the 17-C with 8 to 10 carbon atoms, a yield of 17-C-steroid-.alpha.-propionic acid compounds, measured under standard conditions, of at least 5% by weight, preferably at least 10% by weight, related to the sterol compounds used.
Preferably wild strains of microorganisms are isolated and grown, which when grown on sterol compounds of the above-mentioned type yield a selective splitting according to the general formula: EQU I=a.multidot.10.sup.p
where a denotes the growth factor and p the selective factor of the wild strain growth, and the selectivity index I has a numerical value of at least 1, preferably at least 2, and particularly at least 20. The growth factor a of the wild strain should be at least 0.2 under standard conditions, preferably at least 1, and the selectivity factor p under standard conditions at least 0.5, preferably at least 1. The preferred wild strains for the subsequent production of the defect mutants should prefer the 17-C-side-chain splitting over the ring cleavage. The mutation treatment of the wild strains is effected under such conditions of concentration and a duration of action of the mutagenic agent that 10% to 99.999% of the starting population of the microorganisms are inactivated by the mutagenic treatment, working preferably with a killing rate of 90% to 99.99%.
.DELTA..sup.4 -BNC has the structural formula: ##STR2## and .DELTA..sup.1,4 -BNC has the structural formula: ##STR3##
FIAT final report No. 996 (1947), pages 24-26, describes a chemical modification of phytosterol acetate to progesterone by means of a modified Curtius degradation.
Julian et al, J. Am. Chem. Soc. 70, pp. 887-892 (1948), describes 3-acetoxy-5-bisnor-cholenic acid (BNC) and its conversion to .DELTA..sup.20 -pregnenes by a Curtius procedure.
Moersch et al, J. Org. Chem. 29, pp. 2495-2499 (1964), describe the production of 3-halo-androsta-1,3,5-trienes from androsta-1,4-dien-3-ones.